Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

Abstract

SummaryThe bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membranebound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s -1 . Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s -1 .

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

“…Considering the torque produced by N stators as Nτ 1 = γ2πω N (where τ 1 is the torque produced by a single stator, γ the drag coefficient of the bead, and ω N the measured speed (in Hz) of the motor with N stators), all the strains tested are driven by N ~ 8-10 stators, in line with previous measurements at high load 15,44 . This result supports previous works focused on quantifying steady-state stator stoichiometry by fluorescent stators 15,[18][19][20][21]28,29,[45][46][47] .…”

“…After fusing the fluorescent proteins YPet, eGFP and Dendra2 directly to the N-terminus of MotB (FPs-MotB), as done in several studies with (e) 15,18,19,21,29,38 , we verified that the constructed strains were motile in soft agar at the population level (Fig. 1B).…”

“…The stators dynamically turn over between two populations: one that is bound to the BFM and one that passively diffuses in the inner membrane 15,37 . These dynamics have been observed directly at the single-stator level using fluorescent fusions to the N-terminus of MotB 15,18,19,21,28,29 . More recently, similar fusions have been used to unveil that the number of stators recruited into the complex depend on the load of the BFM 18,19,29 .…”

“…One direction of research for which this holds is the study of the internal dynamics of proteins complexes 13,14 . Recently, several studies have made use of FPFs to study the internal subunit dynamics of the bacterial flagellar motor (BFM) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] . Although FPFs were shown to affect BFM function by decreasing the chemotactic motility of cells and average speed of motors 15 , a quantitative characterization of the impact on the BFM mechanical behavior is lacking.…”

Type III secretion systems: the bacterial flagellum and the injectisome

“…B 370: 20150020 complex, exchanges with an average dwell time of about 30 s [249], and indeed is only maintained in place by a conformational change caused by the ion flow [4,250]. In the E.coli flagellum, there can be up to 11 MotAB stator complexes and their number increases with the external force on the filament [4,[251][252][253][254]. 5 While the PMF is required for the association of stator complexes with the rotor, individual stators only stay associated for about 30 s before exchanging with stators in the membrane, even at full PMF.…”